title: Site-specific glycan-peptide analysis for determination of N-glycoproteome heterogeneity.
abstract: A combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography-tandem mass spectrometry (PGC-LC-MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography (Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC; pH 7.9). The resulting fractions were each separated into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove N-glycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation of a glycopeptide-centric concatenated database for each fraction. The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2.7), employing fragmentation by CID and HCD. The assignment of glycan compositions to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the N-glycan modification database. CID spectra were used to assign glycan structures identified in the glycomic analysis to peptide sequences. This multidimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins.
authors.affiliation: Discipline of Pathology, School of Medical Sciences, The University of Sydney , Sydney 2006, Australia.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Parker
authors.forename: Morten
authors.initials: M
authors.lastname: Thaysen-Andersen
authors.forename: Nestor
authors.initials: N
authors.lastname: Solis
authors.forename: Nichollas E
authors.initials: NE
authors.lastname: Scott
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
authors.forename: Mark E
authors.initials: ME
authors.lastname: Graham
authors.forename: Nicolle H
authors.initials: NH
authors.lastname: Packer
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Cordwell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D016208:Databases, Factual; D018740:Genetic Heterogeneity; D054794:Glycomics / Q000295:instrumentation / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D051381:Rats; D011917:Rats, Inbred Lew; D053719:Tandem Mass Spectrometry
chemical_list: D020543:Proteome; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase